T cell activation is central to initiating an immune response. T cell antigen receptor (TCR) engagement, in concert with costimulatory signals such as through CD28, leads to alterations in actin that control synapse formation, activation of downstream signaling pathways and cytokine production, including IL-2. To identify novel molecules that regulate T cell activation, we established a unique system coupling genetic complementation with Jurkat mutagenesis. Jurkat mutant T cell lines were generated in which TCR/CD28 stimulation failed to activate a reporter containing the RE/AP element from the IL-2 promoter. Biochemical characterization failed to pinpoint the molecular cause for the defects in T cell activation in these cell lines, implying that novel molecules or pathways were responsible. Therefore, we took a genetic approach to rescue the T cell activation defect by retroviral expression of a leukocyte library. From our initial screen, we identified one cell line in which T cell activation was restored due to overexpression of a poorly characterized protein, NKAP. Our work has demonstrated that NKAP is a transcriptional repressor that is required for T cell development and maturation, validating that this approach can identify novel proteins critical for T cell activation. Subsequently, we have identified a second molecule from this screen, COTL1 (coactosin-like protein 1). Coactosin was initially identified in Dictyostelium, as an inhibitor of actin capping, but its function in T cells was unknown. Using shRNA knockdown, we demonstrate that COTL1 is critical for actin remodeling during T cell activation. In the absence of COTL1, Jurkat T cells fail to spread on CD3- coated coverslips or form a lamellipodia when interacting with superantigen-pulsed Raji B cells, consistent with a failure to elongate F-actin fibers. In addition, knockdown of COTL1 blocked TCR/CD28-mediated upregulation of the RE/AP element from the IL-2 promoter, demonstrating that it is required for signal transduction downstream of TCR/CD28 stimulation. Therefore, COTL1 is a novel regulator of actin remodeling and T cell activation. To understand the role of COTL1 in vivo, we have generated mice with a floxed COTL1 allele. The purpose of this R21 is to determine the function of COTL1 in T cell development, activation, F-actin remodeling and migration through the examination of COTL1 conditional knockout mice.